Biological membranes and related systems are being investigated via atomic force microscopy (AFM) and other biophysical technologies in this project containing several collaborations. (1) Collaborating with NIAAA scientists (Drs. B. J. Litman and S.-L. Niu), our OD team is using atomic force microscopy (AFM) to characterize structure and function of the protein rhodopsin, a G-protein coupled receptor (GPCR), in native rod outer segment membranes of the vision pathway, and in reconstituted systems with dipalmitoylphosphatidylcholine (DPPC, di16:0PC) membranes. The AFM imaging has revealed individual rhodopsin molecules at sub-nanometer resolutions as monomers and in various multimeric organizations depending upon the physical state of the membranes and environmental conditions. For biomimicking membranes, we are paying particular attention to the three-component DDPC/DPPC/cholesterol system, where DDPC (di-22:6n-3PC) is polyunsatuatrated and derived from docosahexaenoic acid (DHA). AFM studies of micro- and nano-structures are being combined with thermodynamic information from differential scanning calorimetry (DSC) to explore the connection between rhodopsin signaling and the lipid membrane environment. (2) Collaborating with NIAID scientists (Drs. J.A. Dvorak and F. Tokumasu) and an extramural investigator (Prof. G. W. Feigenson, Cornell Univ.), we have concluded a set of studies on heterogeneities of lipid membranes involving 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), DPPC, dilauroylphosphatidylcholine (DLPC), and cholesterol (chol). We found that biomimicking membranes readily form on atomically flat mica support via large unilamellar vesicle (LUV) surface adsorption and fusion. AFM imaging then revealed extensive nanoscopic membrane domains (c.f. membrane rafts) with diameters around 40 nanometers depending on cholesterol level, DLPC and DPPC ratio, and other phase space parameters such as the strength of mica substrate support. Mathematical analyses were employed in these studies toward a comprehensive understanding of the cellular membrane under influence of its composition and native interactions. Linked to membrane structures and also in collaboration with NIAID scientists (Drs. J.A. Dvorak and T. Arie), we extended our video microscope image analyses of flicker and edge dithering phenomena to the whole cell level to characterize red blood cells during the Plasmodium falciparum malaria infection and development. We found that the parasitic infection markedly modifies cell membrane dynamics in potential relevance to malaria disease mechanism in human microcirculations. (3) Collaborating with another NIAID scientist (Dr. J. Silver), we have developed nanoscopic energetic and structural descriptions of biological membranes to understand fusion pore dynamics important to many cellular processes such as virus invasion and vesicular trafficking. (4) Finally, collaborating with extramural scientists (Drs. A. Sinz and O. Zschornig, Univ. Leipzig, Germany), we have investigated the metal ion binding properties of a fusogenic peptide and its mutants derived from the protein bindin, which is crucial for egg-sperm membrane fusion during sea urchin fertilization. Electrospray Ionization Fourier Transform Ion-Cyclotron Resonance Mass Spectrometry (ESI-FTICRMS) measurements of peptide/metal complexes together with mathematical modeling have added insights on why bindin/Cu2+ suppresses membrane fusion, but bindin/Zn2+ enhances fusion.